Optical imaging of colorectal cancer

ABSTRACT

The invention provides contrast agents for optical imaging of colorectal cancer (CRC) in patients. The contrast agents may be used in diagnosis of CRC, for follow up of progress in disease development, and for follow up of treatment of CRC. Further, the invention provides methods for optical imaging of CRC in patients.

FIELD OF THE INVENTION

The present invention provides contrast agents for optical imaging ofcolorectal cancer (CRC) in patients. The contrast agents may be used indiagnosis of CRC, for follow up of progress in disease development, andfor follow up of treatment of CRC.

The present invention also provides new methods of optical imaging ofCRC in patients, for diagnosis and for follow up of disease developmentand treatment of CRC.

DESCRIPTION OF RELATED ART

Colorectal cancer (CRC) is one of the most frequent malignant diseasesin the Western civilization. More than 100 000 new cases of CRC arediagnosed every year in US and the disease is fatal for a highpercentage of these. CRC is the fourth most commonly diagnosed cancerdisease and it is the second leading cause of cancer death in US afterlung cancer.

The peak incidence of CRC generally occurs after the age of 60 years.CRC is more common in the western world than, in underdevelopedcountries. There may be several reasons for this, including lifeexpectancy, genetic susceptibility and diet. It has been suggested that,intake of fat and red meat has a negative effect on the incidence of CRCwhile a fibre rich diet might decrease the risk for CRC.

Most of the CRC incidents are adenocarcinomas. The sizes of the lesionsare normally in the range from a few millimetres to several centimetres,and the lesions are unevenly distributed througouth the lower part ofthe gastrointestinal system. Normally CRC cells remain superficial for along time and will slowly invade the deeper layers of the intestinalwall and later migrate through the intestinal wall. A majority of thepatients with advanced colorectal cancer develop liver metastasis duringthe course of the disease.

Several therapeutic drugs are today used for treatment of CRC. Theseinclude Eloxatine® (oxaliplatin), Camptosar® (irinotecan), OncoVAX,Tomudex® (raltitrexed), TS-1, Futulon (doxifluridine) and Xeloda(capecitabine). Several therapeutic products are in late developmentincluding Thalomide® (thalidomide), Avastin® (bevacizumab), NeuTrexin®(trimetrexate), Panorex® (edrecolomab) and Erbitux (cetuximab).

The prognosis for the patient is very dependent on the progress of thedisease. With no metastasis and localization of the tumor(s) to bowelmucosa the 5-year survival prognosis is 80%, while patients withadvanced CRC with distant metastasis have a low (<5%) 5-year survivalprognosis.

Accordingly, it is critical to diagnose CRC at an early stage before thedisease invades deeper layers of the intestinal wall and before thepatients develop liver metastasis. The clinical symptoms of CRC areoften non-specific. However, typical symptoms can be discoloured stool(blood in the stool), abdominal pain, weight loss, fever and diarrhoea.The methods used to diagnose CRC include coloscopy, fecal occult bloodtesting, sigmoidoscopy and double-contrast barium colonography. CTcolonography is comparable to colonoscopy for detection of colorectalpolyps equal to or larger than 10 mm. The American Cancer Society andothers have suggested performing CRC screening of the population orparts of the population. Several clinical studies conclude thatscreening for CRC is cost effective compared to no screening. Althoughscreening methods for early detection of CRC is available, many patientshave CRC diagnosed at a late stage and have poor prognosis. There areseveral advantages related to the methods used to screen and diagnoseCRC today. However, colonoscopy has always a risk of perforation, faecaloccult blood testing results in very many false positive results basedon other sources of blood, as for example haemorrhoids. No methods,including x-ray methods, are CRC specific and therefore result in manyfalse positive results (e.g. polyps). Existing diagnostic methods fordiagnosis of CRC not only result in many false positive results, but theuse of these methods also results in many false negative results. Noneof these methods are useful for safe early diagnosis of CRC at the stagewhere the disease is superficial. The most specific method might bepositron emission tomography (PET) with fluorodeoxyglucose (FDG), butthis method is expensive and should be reserved for equivocal cases.

A study of recently published literature on CRC shows that there is amedical need for a cheap, simple and safe method for diagnosis of CRC atan early stage.

U.S. Pat. No. 6,455,688 claims a method for diagnosing CRC bydetermining the expression of a gene encoding a specific sequence(CJA8).

U.S. Pat. No. 6,326,148 provides a method of screening for coloncarcinoma cells in a sample by determining the presence of increasedcopy number of chromosome 20q.

U.S. Pat. No. 6,316,272 suggests a method of diagnosis of CRC related toa specific nucleic acid sequence.

U.S. Pat. No. 6,187,591 claims a screening test for colorectal cancerwhereby a marker is detected in rectal mucus. The marker is detected inthe mucus deposited on a support using Schiff's reagent.

U.S. Pat. No. 6,150,100 claims a method for diagnosis of tumors of thegastrointestinal tract such as colorectal tumors based on determinationof the genomic instability at 5 selected microsatelite loci.

U.S. Pat. No. 6,149,581 claims a device and method for access to thecolon and small bowel of a patient.

U.S. Pat. No. 5,416,025 claims a method for detecting CRC by adding anenzyme to a mucus sample to detect a specific disaccharide marker.

U.S. Pat. No. 5,380,647 claims a test for detecting carcinoembryonicantigen (CEA) in stool. CEA is indicator of the presence of CRC.

U.S. Pat. No. 4,996,298 claims a new method for diagnosis of CRC basedon glycoprotein as a marker for CRC.

U.S. Pat. No. 4,857,457 claims a method for detecting the presence ofprecancer or cancer of the large intestine by assaying the presence of adisaccharide in a mucus sample.

JP II-225800 claims a method for detecting colon cancer usingfluorescent material. The method relates to telomerase, however, themethod is an in vitro method and does not suggest contrast agents.

As pointed out CRC is still a challenge to diagnose and treat. There isa need for improved diagnostic methods, especially for diagnosis of CRCin an early stage with good reliability. We have surprisingly discoveredthat the use of optical imaging methods and new contrast agents fulfillthese requirements.

SUMMARY OF THE INVENTION

In view of the needs of the art the present invention provides anoptical imaging contrast agent with affinity for an abnormally expressedbiological target associated with CRC.

The invention is also described in the claims.

The following definitions will be used throughout the document:

CRC tissue: Any tissue in the colon or rectum that shows changesassociated with neoplasia or preneoplasia, and including metastases fromcolorectal cancer at other sites in the body.

Abnormally expressed target: A target that is either overexpressed,downregulated or mutated in CRC tissue.

Overexpressed target: A receptor, an enzyme or another molecule orchemical entity that is present in a higher amount in CRC tissue than innormal tissue.

Downregulated target: A receptor, an enzyme or another molecule orchemical entity that is present in a lower amount in CRC tissue than innormal tissue.

Mutated target: A protein in CRC tissue that is altered as a result of agermline or sotatic mutation, and including alterations resulting fromdifferential splicing of RNA and changes in post-translationalmodifications, particularly glycosylation patterns, but not limitedto-these types of alterations.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is an optical imaging contrastagents for imaging of CRC. By the term optical imaging contrast agent,or just contrast agent, we mean a molecular moiety used for enhancementof image contrast in vivo comprising at least one moiety that interactswith light in the ultraviolet, visible or near infrared part of theelectromagnetic spectrum.

The contrast agent has affinity for an abnormally expressed targetassociated with CRC. By abnormally expressed, is meant that the targetis either downregulated, mutated or overexpressed. That is, the contrastagent has affinity for a target that is either downregulated, mutated oroverexpressed in CRC tissue.

CRC tissue containing a downregulated target is identified by a lowamount of bound contrast agent compared to normal tissue. In thissituation, the amount of contrast agent should be less than 50% of thatin normal tissue, preferably less than 10%.

Targets that are mutated in CRC tissue are identified by lack of bindingof a contrast agent that does bind to normal tissue; alternatively, thecontrast agent might be directed specifically towards the mutatedtarget, and binding to normal tissue would be minimal. Mutations inCRC-associated genes are often non-random. For instance, more than 90%of mutations in the K-ras gene observed in CRC occur at codon 12 or 13.Somatic mutations in the important adenomatous polyposis coli (APC) genecommonly occur at codons 1309-1311 or codon 1450.

Preferred contrast agents according to the invention have affinity foran overexpressed target associated with CRC. Preferred targets are thosetargets that are more than 50% more abundant In CRC tissue than insurrounding tissue. More preferred targets are those targets that aremore than two times more abundant in CRC tissue than in surroundingtissue. The most preferred targets are those targets that are more than5 times more abundant in CRC tissue than in surrounding tissue.

Relevant groups of targets are receptors, enzymes, nucleic acids,proteins, lipids, other macromolecules as for example lipoproteins andglycoproteins. The targets may be located in the vascular system, in theextracellular space, associated with cell membranes or locatedintracellularly.

The following biological targets are preferred targets for contrastagents for optical imaging of CRC:

Adhesion Molecules and Adhesion-Associated Molecules:

Beta-catenin, E-cadherin (CDH1 gene), adenomatous polyposis coli protein(APC), p120-catenin, CD44-standard, CD44-6v, CD44-9v, 67-kDa lamininreceptor, P-cadherin and integrins, such as α_(v)β₃. and α_(v)β₆.

Antigens:

Human leukocyte antigen-B18 and human leukocyte antigen-DQ5, tissuepolypeptide antigen (TPA) or tissue polypeptide-specific antigen (TPS),Small intestinal mucin antigen (SIMA), CA15.3, CA 19-9, CA 72-4, CYFRA21-1, CAM 17.1, CEA, TPS, CA 72-4, MUC-1, tumour-associated antigen L6,HLA-A, CA-195, CA-242, beta HCG, AFP, CA125.

Enzymes:

alpha-methylacyl-CoA racemase, aminopeptidase N/CD13, carcinogenmetabolising enzymes, arachidonic acid metabolism, enzymes responsiblefor polyamine metabolism, CDC25B phosphatase, COX-1, cyclooxygenase-2(COX-2), cytochrome P450 2A6 (CYP2A6), glutathione S-transferase,gamma-glutamylcysteine synthetase (gamma-GCS) and DT-diaphorase,guanylyl cyclase C, matrix metalloproteinases and their inhibitors(especially MMP-2, MMP-7, MMP-9, stromelysin-3 and MT1-MMP (=MMP 14)),mitochondrial aspartate-aminotransferase, phosphoglucomutase,plasminogen-related molecules, thymidylate synthase, tumour-associatedtrypsin inhibitor (TATI), u-PA, prostaglandin E synthase and cathepsins,typically cathepsin B and human aspartyl (asparginyl) beta-hydroxylase(HAAH),

Signal Molecules and Receptors:

Beta-HCG, c-erbB, and VEGF, c-Myc, gastrin, CCK(B)-R, gastrin, bradeion(septin family gene), WNT7A, WNT7B, insulin-like growth factor 2,benzodiazepine receptor, Her-2, VEGF receptors, EGF receptors, IL-8,CXCR1, CXCR2, Urokinase Plasminogen Activator Receptor (u-PAR),urokinase receptor associated protein (u-PARAP/Endo 180), c-met andangiotensin1 -receptor (AT1R).

Tumour Suppressor Proteins, Oncogenes, Apoptosis-Related Proteins:

Adenomatous polyposis coli protein (APC), bax, Bcl-2, beta-catenin/Tcell factor-4 (Tcf-4), groucho proteins, proteins in K-ras cascade,nm23, p53, K-ras, Deleted in Colorectal Cancer (DCC), c-erbB2, survivin,SMAD2, SMAD4.

Others:

L-plastin, the human homologue of yeast ribosomal protein S28, theB-cell translocation gene, AXIN2, chromogranin A, synaptophysin,syntaxin1, VAMP2, SNAP25, alpha/beta-SNAP, clusterin (apolipoprotein j),ITF-2, PPARdelta, cystatin-like metastasis-associated protein, EBP50,etheno (epsilon)-DNA adducts (e.g., via trans-4-hydroxy-2-nonenal),keratin 5, Ki-67, Mib-1, proliferating cell nuclear antigen,osteopontin, p27 (kip-1), proliferating cell nuclear antigen (PCNA),WAF1, p34cdc2, cyclins B1 and D1, SBA2, sigma B3 protein, transcriptionfactor nuclear factor-kappa beta (NF-kappaB) and hypoxia-induciblefactor.

Among the more preferred targets for contrast agents for optical imagingof CRC are: COX-2, beta-catenin, E-cadherin, P-cadherin, variouskinases, Her-2, MMPs, cyclins, P53, thymidylate synthase, VEGFreceptors, EGF receptors, K-ras, adenomatous polyposis coli protein,cathepsin B, uPAR, c-met, mucins and gastrin receptors.

The most preferred targets for contrast agents for optical imaging ofCRC are: c-met, MMP-14, COX-2, beta-catenin and cathepsin B.

Generally, any targets that have been identified as possible targets foragents for treatment of CRC are potential targets also in opticalimaging.

The preferred contrast agents are molecules with relatively lowmolecular weights. The molecular weight of preferred contrast agents isbelow 10000 Daltons, more preferably below 7000 Daltons.

The contrast agents are preferably comprised of a vector that hasaffinity to an abnormally expressed target in CRC tissue, and an opticalreporter. Thus viewed from one aspect the present invention provides acontrast agent of formula I:V-L-R   (I)

wherein V is one or more vector moieties having affinity for one or moreabnormally expressed target in CRC tissue, L is a linker moiety or abond and R is one or more reporter moieties detectable in opticalimaging.

The vector has the ability to direct the contrast agent to a region ofCRC. The vector has affinity for the abnormally expressed target andpreferably binds to the target. The reporter is detectable in an opticalimaging procedure and the linker must couple vector to reporter, atleast until the reporter has been delivered to the region of CRC andpreferably until the imaging procedure has been completed.

The vector can generally be any type of molecules that have affinity forthe abnormally expressed target. The molecules should be physiologicallyacceptable and should preferably have an acceptable degree of stability.The vectors can for instance be selected from the following group ofcompounds: peptides, peptoids/peptidomimetics, oligonucleotides,oligosaccharides, lipid-related compounds like fatty acids, traditionalorganic drug-like small molecules, synthetic or semi-synthetic, andderivatives and mimetics thereof. When the target is an enzyme thevector may comprise an inhibitor of the enzyme or an enzyme substrate.The vector of the contrast agent preferably has a molecular weight ofless than 4500 Daltons and more preferably less than 2500 Daltons.

Contrast agents having affinity for more than one abnormally expressedtarget related to the disease is an aspect of the invention. Suchcontrast agents can comprise two or more different vectors or molecularsubunits that target two or more different abnormally expressed targets.

Another possibility according to the present invention is that thecontrast agent comprises one vector that is able to bind to more thanone abnormally expressed target in CRC tissue.

A contrast agent according to the present invention can also comprisemore than one vector of same chemical composition that bind to theabnormally expressed biological target.

Some receptors are unique to endothelial cells and surrounding tissues.Examples of such receptors include growth factor receptors such as VEGFand adhesion receptors such as the integrin family of receptors.Peptides comprising the sequence arginine-glycine-aspartic acid (RGD)are known to bind to a range of integrin receptors. Such RGD-typepeptides constitute one group of vectors for targets associated withCRC.

Below are some examples of vectors having affinity for CRC relatedabnormally expressed targets:

Vectors for COX-2:

Arachidonic acid is the endogenous substrate for COX-2.

Other vectors for COX-2 are exogenous compounds that bind to COX-2, forexample so-called COX-2 inhibitors. The chemical classes of the mainCOX-2 inhibitors are shown in WO 02/07721.

Vectors for MMP-7:

Peptide sequence: Cys-Gly-Pro-Leu-Gly-Leu-Leu-Ala-Arg-OH

Vector for Benzodiazepine Receptor:

Vectors for Integrins: RGD-Type Peptides Having Affinity for α_(v)β₃.and α_(v)β₅:

c[-Asp-D-Phe-Lys-Arg-Gly-]

A wide variety of linkers can be used. The linker component of thecontrast agent is at its simplest a bond between the vector and thereporter moieties. In this aspect the reporter part of the molecule isdirectly bound to the molecule sub-unit that binds to the abnormallyexpressed target. More generally, however, the linker will provide amono- or multi-molecular skeleton covalently or non-covalently linkingone or more vectors to one or more reporters, e.g. a linear, cyclic,branched or reticulate molecular skeleton, or a molecular aggregate,with in-built or pendant groups which bind covalently or non-covalently,e.g. coordinatively, with the vector and reporter moieties. The linkergroup can be relatively large in order to build into the contrast agentoptimal size or optimal shape or simply to improve the bindingcharacteristics for the contrast agent to the abnormally expressedtarget in CRC tissue.

Thus, linking of a reporter unit to a desired vector may be achieved bycovalent or non-covalent means, usually involving interaction with oneor more functional groups located on the reporter and/or vector.Examples of chemically reactive functional groups which may be employedfor this purpose include amino, hydroxyl, sulfhydroxyl, carboxyl andcarbonyl groups, as well as carbohydrate groups, vicinal diols,thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl andphenolic groups.

The reporter is any moiety capable of detection either directly orindirectly in an optical imaging procedure. The reporter might be alight scatterer (e.g. a coloured or uncoloured particle), a lightabsorber or a light emitter. More preferably the reporter is a dye suchas a chromophore or a fluorescent compound. The dye part of the contrastagent can be any dye that interacts with light in the electromagneticspectrum with wavelengths from the ultraviolet light to the nearinfrared. Preferably, the contrast agent of the invention hasfluorescent properties.

Preferred organic chromophoric and fluorophoric reporters include groupshaving an extensive delocalized electron system, eg. cyanines,merocyanines, indocyanines, phthalocyanines, naphthalocyanines,triphenylmethines, porphyrins, pyrilium. dyes, thiapyrilium dyes,squarylium dyes, croconium dyes, azulenium dyes, indoanilines,benzophenoxazinium dyes, benzothiaphenothiazinium dyes, anthraquinones,napthoquinones, indathrenes, phthaloylacridones, trisphenoquinones, azodyes, intramolecular and intermolecular charge-transfer dyes and dyecomplexes, tropones, tetrazines, bis(dithiolene) complexes,bis(benzene-dithiolate) complexes, iodoaniline dyes, bis(S,O-dithiolene)complexes. Fluorescent proteins, such as green fluorescent protein (GFP)and modifications of GFP that have different absorption/emissionproperties are also useful. Complexes of certain rare earth metals(e.g., europium, samarium, terbium or dysprosium) are used in certaincontexts, as are fluorescent nanocrystals (quantum dots).

Particular examples of chromophores which may be used includefluorescein, sulforhodamine 101 (Texas Red), rhodamine B, rhodamine 6G,rhodamine 19, indocyanine green, Cy2, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7,Cy7.5, Marina Blue, Pacific Blue, Oregon Green 488, Oregon Green 514,tetramethylrhodamine, and Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680,Alexa Fluor 700, and Alexa Fluor 750. The cyanine dyes are particularlypreferred.

Particularly preferred are dyes which have absorption maxima in thevisible or near infrared region, between 400 nm and 3 μm, particularlybetween 600 and 1300 nm. The contrast agents according to the inventioncan comprise more than one dye molecular sub-unit. These dye sub-unitscan be similar or different from a chemical point of view. Preferredcontrast agents have less than 6 dye molecular sub-units.

Several relevant targets for CRC are enzymes. A contrast agent foroptical imaging of CRC for targeting an enzyme can be an enzyme contrastagent substrate that can be transformed to a contrast agent productpossessing different pharmacokinetic and/or pharmacodynamic propertiesfrom the contrast agent substrate. This embodiment of the inventionprovides contrast agent substrates having affinity for an abnormallyexpressed enzyme,.wherein the contrast agent substrate changespharmacodynamic and/or pharmacokinetic properties upon a chemicalmodification into a contrast agent product in a specific enzymatictransformation, and thereby enabling detection of areas of disease upona deviation in the enzyme activity from the normal. Typical differencesin pharmacodynamic and/or pharmacokinetic properties can be bindingproperties to specific tissue, membrane penetration properties, proteinbinding and solubility properties.

Alternatively, if the abnormally expressed target for diagnosis of CRCis an enzyme, the contrast agent for optical imaging can be a dyemolecule that directly binds to the enzyme. The contrast agent will haveaffinity for the abnormally expressed enzyme, and this may be used toidentify tissue or cells with increased enzymatic activity.

In a further aspect of the invention, the contrast agent changes dyecharacteristics as a result of an enzymatic transformation. For example,a fluorescent dye reporter of the contrast agent is quenched (nofluorescence) by associated quencher groups, until an enzymatic cleavagetakes place, separating the dye from the quencher groups and resultingin fluorescence at the site of the abnormally expressed enzyme.

Another aspect of this part of the invention is that the dye may changecolour, as e.g. a change in absorption and/or emission spectrum, as aresult of an enzymatic transformation.

If the abnormally expressed target for diagnosis of CRC is a receptor oranother non-catalytical target, the contrast agent for optical imagingcan bind directly to the target and normally not change the dyecharacteristics.

Another aspect of the invention is contrast agents for optical imagingof CRC characterized by having affinity for more than one abnormallyexpressed target related to the disease. Such contrast agents can havetwo or more different vectors or molecular subunits that target two ormore different abnormally expressed targets.

The preferred contrast agents of the present invention are soluble inwater. This means that the preferred contrast agents have a solubilityin water at pH 7.4 of at least 1 mg/ml.

The contrast agents of the present invention can be identified by randomscreening, for example by testing of affinity for abnormally expressedtargets of a library of dye labelled compounds either prepared andtested as single compounds or by preparation and testing of mixture ofcompounds (a combinatorial approach).

The contrast agents of the present invention can also be identified byuse of technology within the field of intelligent drug design. One wayto perform this is to use computer-based techniques (molecular modellingor other forms of computer-aided drug design) or use of knowledge aboutnatural and exogenous ligands (vectors) for the abnormally expressedtargets. The sources for exogenous ligands can for example be thechemical structures of therapeutic molecules for targeting the sametarget. One typical approach here will be to bind the dye chemicalsub-unit to the targeting vector so that the binding properties of thevector are not reduced. This can be performed by linking the dye at thefar end away from the pharmacophore centre (the active targeting part ofthe molecule). Alternatively, random screening may be used to identifysuitable vectors before labelling with a reporter.

The contrast agents of the invention are preferably not endogenoussubstances alone. Some endogenous substances, for instance estrogen,have certain fluorescent properties in themselves, but they are notlikely to be sufficient for use in optical imaging. Endogenoussubstances combined with an optical reporter however, falls within thecontrast agents of the invention.

The contrast agents of the invention are intended for use in opticalimaging. Any method that forms an image for diagnosis of disease, followup of disease development or for follow up of disease treatment based oninteraction with light in the electromagnetic spectrum from ultravioletto near-infrared radiation falls within the term optical imaging.Optical imaging further includes all methods from direct visualizationwithout use of any device and use of devices such as various scopes,catheters and optical imaging equipment, for example computer basedhardware for tomographic presentations. The contrast agents will beuseful with optical imaging modalities and measurement techniquesincluding, but not limited to: luminescence imaging; endoscopy;fluorescence endoscopy; optical coherence tomography; transmittanceimaging; time resolved transmittance imaging; confocal imaging;nonlinear microscopy; photoacoustic imaging; acousto-optical imaging;spectroscopy; reflectance spectroscopy; interferometry; coherenceinterferometry; diffuse optical tomography and fluorescence mediateddiffuse optical tomography (continuous wave, time domain and frequencydomain systems), and measurement of light scattering, absorption,polarisation, luminescence, fluorescence lifetime, quantum yield, andquenching.

Examples of contrast agents for optical imaging of CRC according to theinvention are shown below:

Contrast Agents for Mapping of COX-2:

1.

Wherein arachidonic acid, the endogenous substrate for COX-2, is coupledto a reporter (R) via a linker (L).

2.

Wherein a COX-2 inhibitor derivative is linked to a reporter. R is anyreporter according to the present invention; for example fluorescein,and L is a linker.

Contrast Agent for Mapping of Matrix Metalloproteinase:

The vector peptide is linked to e.g. fluorescein (R) through a linker(L):

Contrast Agent with Affinity for Benzodiazepine Receptor:

Wherein L is a linker and R is one of the mentioned reporters.

A further embodiment is use of contrast agents of the invention foroptical imaging of CRC, that is, for diagnosis of CRC, for use in followup the progress in CRC development or for follow up the treatment ofCRC. In the context of this invention, diagnosis includes screening ofselected populations, early detection, biopsy guidance,characterisation, staging, grading, therapy efficacy monitoring,long-term follow-up of relapse and surgical guidance.

Still another embodiment of the invention is a method of optical imagingfor diagnosis of CRC using the contrast agents as described.

Still another embodiment of the invention is a method of optical imagingfor diagnosis, to follow up the progress of CRC development and tofollow up the treatment of CRC, using the contrast agents as described.

One aspect of these methods is to administer the present contrast agentsand follow the accumulation and elimination directly visually duringsurgery. Another aspect of these methods is to administer the presentcontrast agents and perform visual diagnosis through a colonoscope.

Still another aspect of the present invention is to administer thepresent contrast agents and perform the image diagnosis usingcomputerized equipment as for example a tomograph.

Still another embodiment of the invention is use of a contrast agent asdescribed for the manufacture of a diagnostic agent for use in a methodof optical imaging of CRC involving administration of said diagnosticagent to an animate body and generation of an image of at least part ofsaid body.

Still another embodiment of the invention is pharmaceutical compositionscomprising one or more contrast agents as described or pharmaceuticallyacceptable salts thereof for optical imaging for diagnosis of CRC, forfollow up progress of CRC development or for follow up the treatment ofCRC. The diagnostic agents of the present invention may be formulated inconventional pharmaceutical or veterinary parenteral administrationforms, e.g. suspensions, dispersions, etc., for example in an aqueousvehicle such as water for injections. Such compositions may furthercontain pharmaceutically acceptable diluents and excipients andformulation aids, for example stabilizers, antioxidants, osmolalityadjusting agents, buffers, pH adjusting agents, etc. The most preferredformulation is a sterile solution for intravascular administration orfor direct injection into area of interest. Where the agent isformulated in a ready-to-use form for parenteral administration, thecarrier medium is preferably isotonic or somewhat hypertonic.

The dosage of the optical diagnostic agents of the invention will dependupon the clinical indication, choice of contrast agent and method ofadministration. In general, however dosages will be between 10 μg and 5grams for an adult human;

While the present invention is particularly suitable for methodsinvolving parenteral administration of the contrast agent, e.g. into thevasculature or directly into an organ of muscle tissue, intravenousadministration being especially preferred, it is also applicable whereadministration is not via a parenteral route, e.g. where administrationis transdermal, nasal, sub-lingual or is into an externally voiding bodycavity, e.g. the colon, rectum or bladder. The present invention isdeemed to extend to cover such administration.

The following examples are illustrative only and not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description and from the claims.

EXAMPLES Example 1 Contrast Agent for Mapping of COX-2 Activity.Synthesis of COX-2 Ligand Coupled to Fluorescein

Step 1

2-Hydroxy-1-(4-methanesulfonylphenyl)ethanone is prepared from2-bromo-1-(4-methanosulfonylphenyl)ethanone according to C. Puig et alin J. Med. Chem 2000,43, 214-223.

Step 2

A solution of 2-hydroxy-1-(4-methanosulfonylphenyl)ethanone (1.50 g, 7mmol) and fluorescein isocyanate isomer 1 (2.72 g, 7 mmol) is heated inDMF at 120° C. for 5 hours.

The mixture is cooled, DMF evaporated off and acetic acid (25 ml) isadded. The mixture is refluxed for 10 hours. The acetic acid isevaporated and the resulting mixture is purified on silica usingchloroform/methanol as eluent.

Example 2 Contrast Agent for Mapping of Matrix Metalloproteinase (MMP).Synthesis of fluorescein-Cys-Gly-Pro-Leu-Gly-Leu-Leu-Ala-Arg-OH LinkerConjugate

Step 1

The peptide component was synthesised on an ABI 433A automatic peptidesynthesiser starting with Fmoc—Arg(Pmc)—wang resin on a 0.1 mmol scaleusing 1 mmol amino acid cartridges. The amino acids were pre-activatedusing HBTU before coupling. An aliquot of the peptide resin was thentransferred to a clean round bottom flask an N-methyl morpholine (1mmol) in DMF (5 ml) added followed by chloroacetyl chloride (1 mmol).The mixture was gently shaken until Kaiser test negative. The resin wasextensively washed with DMF.

Step 2

5(6)—carboxyfluorescein (188 mg, 0.5 mmol) and dicyclohexylcarbodiimide(113 mg, 0.55 mmol) are dissolved in DMF (20 ml). The mixture is stirredfor 2 hours and cooled to 0° C. A solution of hexamethylenediamide (116mg, 1 mmol) and DMAP (30 mg) in DMF is added and the mixture is stirredat ambient temperature for 72 hours. The solution is evaporated and theconjugate between carboxyfluorescein and hexamethylene-amine is isolatedas monoamide by chromatography (silica, chloroform and methanol).

Step 3

The resin from step 1 is suspended in DMF (5 ml) and amide-amineconjugate from step 2 (0.5 mmol) pre-dissolved in DMF (5 ml) containingtriethylamine (0.5 mmol) is added. The mixture is heated to 50° C. for16 hours then excess reagents filtered off, following extensive washingwith DMF, DCM and diethyl ether then air drying. The product is treatedwith TFA containing TIS (5%), H_(z)0 (5%), and phenol (2.5%) for 2hours.

Excess TFA is removed in vacuo and the peptide is precipitated by theaddition of diethyl ether. The crude peptide conjugate is purified bypreparative HPLC C C-18, acetonitril, TFA, water).

Example 3 Contrast Agent for Binding to Benzodiazepine Receptor

Step 1

Nitrazepam is reduced to the corresponding 7-aminonitrazepam usingstandard condition zink in aqueous hydrochloric acid, catalytichydrogenation or other reduction agents.

Step 2

5(6) Carboxyfluorescein (1 mmol) and dicyclohexylcarbodiimide (1 mmol)are dissolved in DMF (30 ml). The mixture is stirred for 2 hours atambient temperature. A solution of 7-aminonitrazepam (1 mmol) and DMAP(20 mg) in DMF (10 ml) is added and the mixture is evaporated and theconjugate between 7-aminonitrazepam and 5(6) carboxyfluorescein isisolated by chromatography (silica, chloroform/methanol).

Example 4 Contrast Agent for Binding to p53 Oncoprotein

Step 1 Synthesis of2,2-bis(hydroxymethyl)-1-aza-bicyclo[2,2,2,]octan-3-one.3-quinuclidinone hydrochloride (Aldrich Q 190-5) (1 mmol) is dissolvedin methanol-water (1:1, 30 ml). An aqueous solution of formaldehyde(37%, 2.5 mmol) and sodium hydroxide (1.5 mmol) are added. The mixtureis stirred for 12 hours at 50° C. The solvents are evaporated and thetitle compound isolated as free base using flash chromatography (silica,ethylacetate/chloroform, hexane).

Step 2.

5(6)-carboxyfluorescein (0.1 mmol) and dicyclohexyl carbodiimide (0.11mmol) are dissolved in DMF. The mixture is stirred for 3 hours andcooled to 0° C. A solution of2,2-bis(hydrozymethyl)-1-azabicyclo[2,2,2]octane-3-one (0.5 mmol) andDMAP (10 mg) in DMF is added and the mixture is stirred at ambienttemperature for 72 hours. The solution is evaporated and the contrastagent is isolate by flash chromatography (silica, ethyl acetate/hexane).

Example 5 Contrast Agent with Affinity for Integrins: RGD Peptide Linkedto Cy5.5

Step 1. Assembly of Amino Acids

The peptide sequence Asp-D-Phe-Lys-Arg-Gly was assembled on an AppliedBiosystems 433A peptide synthesizer starting with 0.25 mmolFmoc-Gly-SASRIN resin. An excess of 1 mmol pre-activated amino acids(using HBTU; O-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosohate) was applied in the coupling steps. The cleavage ofthe fully protected peptide from the resins was carried out by treatmentof the resin with three portions of 35 mL of 1% trifluoroacetic acid(TFA) in dichloromethane (DCM) for 5 minutes each. The filtratescontaining the peptide was immediately neutralised with 2% piperidine inDCM. The organics were extracted with water (3×100 mL), dried with MgSO₄and evaporated in vacuo. Diethyl ether was added to the residue and theprecipitate washed with ether and air-dried affording 30 mg of crudeprotected peptide. The product was analysed by analytical HPLC(conditions: Gradient, 20-70% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; flow, 2 mL/min; column, Phenomenex Luna 3μ 5×4.6 mm;detection, UV 214 nm; product retention time 7.58 min). Further productcharacterisation was carried out using electrospray mass spectrometry(MH⁺ calculated, 1044.5; MH⁺ found, 1044.4).

Step 2. N-C Cyclisation

30 mg of the fully protected peptide, 16 mg of PyAOP, 4 mg of HOAt and 6μL of N-methylmorpholine (NMM) were dissolved in dimethylformamide/DCM(1:1) and stirred over night. The mixture was evaporated in vacuo anddiethyl ether added to the residue. The precipitate was washed withether and air-dried. The crude cyclic fully protected peptide wastreated with a solution of 25 mL TFA containing 5% water, 5%triisopropylsilane and 2.5% phenol for two hours. TFA was evaporated invacuo and diethyl ether added to the residue. The precipitate was washedwith ether and air-dried. Purification by preparative RP-HPLC (0-30% Bover 40 min, where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, at a flow rateof 10 mL/min on a Phenomenex Luna 5μ C18 250×21.20 mm column) of thecrude material afforded 2.3 mg pure product peptide. The pure productwas analysed by analytical HPLC (conditions: Gradient, 0-15% B over 10min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA; flow, 2 mL/min; column,Phenomenex Luna 3μ 5×4.6 mm; detection, UV 214 nm; product retentiontime 6.97 min). Further product characterisation was carried out usingelectrospray mass spectrometry (MH⁺ calculated, 604.3; MH⁺ found,604.4).

Step 3. Conjugation of Cy5.5 to RGD Peptide

0.6 mg of the RGD peptide, 1:7 mg of Cy5.5 mono NHS ester and 5 μL ofNMM were dissolved in 1 mL of dimethylformamide (DMF) and the reactionmixture stirred for 2 hrs. Diethyl ether was added to the DMF solutionand the blue precipitate washed with diethyl ether and air-driedaffording 0.7 mg of crude RGD peptide conjugated to Cy5.5.The pureproduct was analysed by analytical HPLC (conditions: Gradient, 5-50 % Bover 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA; flow, 0.3 mL/min;column, Phenomenex Luna 3μ5×2 mm; detection, UV 214 nm; productretention time 8.32 min). Further product characterisation was carriedout using electrospray mass spectrometry (MH⁺ calculated, 1502.5; MH⁺found, 1502.6).

Example 6 Synthesis of Losartan Derivatised with Cy5.5—Contrast Agentfor Binding to AT1R

a) Replacement of Losartan Hydroxyl Group by Azide

To a stirred suspension of Losartan (MSD, 0.423 g, 1.00 mmol) anddiphenylphosphoryl azide (Aldrich, 0.259 ml, 1.20 mmol) intetrahydrofuran (8 ml) was added DBU (0.329 ml, 2.20 mmol). Afterstirring overnight water/acetonitrile (1:1, 4.8 ml) was added and themixture was filtered. After addition of neat TFA (to pH 2) the mixturewas purified by preparative HPLC (column Phenomenex Luna C18(2) 5 μm21.2×250 mm, solvents: A=water/0.1% TFA and B=acetonitrile/0.1% TFA;gradient 35-45% B over 60 min; flow 10.0 ml/min, UV detection at 214 nm)in several runs to give.99 mg (22%) of the product as white crystalsafter lyophilisation. Analysis by LC-MS (column Phenomenex Luna C18(2) 3μm 50×4.60 mm, solvents: A=water/0.1% TFA and B=acetonitrile/0.1% TFA;gradient 20-80% B over 10 min; flow 1 ml/min, UV detection at 214 nm,ESI-MS) gave a peak at 7.3 minutes with m/z 448.1 (MH⁺) corresponding tothe structure.

b) Reduction of the Azide Group to Amino Function

To a solution of compound from a) (5.0 mg, 0.011 mmol) in methanol (3ml) was added Pd/C (Koch-Light, ca 10 mg). The mixture was stirred underhydrogen (1 atm) for 10 min, filtered and concentrated. The residue wasused in the next step without further work up. LC-MS analysis (columnPhenomenex Luna C18(2) 3 μm 50×4.60 mm, solvents: A=water/0.1% TFA andB=acetonitrile/0.1% TFA; gradient 20-80% B over 10 min; flow 1 ml/min,UV detection at 214 nm, ESI-MS) gave a peak at 1.9 minutes with m/z422.2 (MH⁺) corresponding to the amine.

c) Conjugation of Cy5.5

To a solution of losartanamine from b) (0.5 mg, 1 μmol) in DMF (0.2 ml)were added Cy5.5-NHS (Amersham Biosciences; 1 mg, 1 μmol) andN-methylmorpholine (1 μl, 9 μmol). The reaction mixture was stirredovernight Formation of losartan-Cy5.5 conjugate was confirmed by MSanalysis giving m/z at 1320.6 (M⁺), expected 1320.3.

Example 7 Synthesis of3-[(4′-Fluorobiphenyl-4-sulfonyl)-(1-hydroxycarbamoylcyclopentyl)amino]Propionicacid (compound A) Derivatised with Cy5.5—Contrast Agent for Binding toMMP

a) 1.11-Diazido-3,6,9-trioxaundecane

A solution of dry tetraethylene glycol (19.4 g, 0.100 mol) andmethanesulphonyl chloride (25.2 g, 0.220 mol) in dry THF (100 ml) waskept under argon and cooled to 0° C. in an ice/water bath. To the flaskwas added a solution of triethylamine (22.6 g, 0.220 mol) in dry THF (25ml) dropwise over 45 min. After 1 hr the cooling bath was removed andstirring was continued for 4 hrs. Water (60 ml) was added. To themixture was added sodium hydrogencarbonate (6 g, to pH 8) and sodiumazide (14.3 g, 0.220 mmol), in that order. THF was removed bydistillation and the aqueous solution was refluxed for 24 h (two layersformed). The mixture was cooled and ether (100 ml) was added. Theaqueous phase was saturated with sodium chloride. The phases wereseparated and the aqueous phase was extracted with ether (4×50 ml).Combined organic phases were washed with brine (2×50 ml) and dried(MgSO₄). Filtration and concentration gave 22.1 g (91%) of yellow oil.The product was used in the next step without further purification.

b) 11-Azido-3,6,9-trioxaundecanamine

To a mechanically, vigorously stirred suspension of1,11-diazido-3,6,9-trioxaundecane (20.8 g, 0.085 mol) in 5% hydrochloricacid (200 ml) was added a solution of triphenylphosphine (19.9 g, 0.073mol) in ether (150 ml) over 3 hrs at room temperature. The reactionmixture was stirred for additional 24 hrs. The phases were separated andthe aqueous phase was extracted with dichloromethane (3×40 ml). Theaqueous phase was cooled in an ice/water bath and pH was adjusted to ca12 by addition of KOH. The product was extracted into dichloromethane(5×50 ml). Combined organic phases were dried (MgSO₄). Filtration andevaporation gave 14.0 g (88%) of yellow oil. Analysis by MALDI-TOF massspectroscopy (matrix: □-cyano-4-hydroxycinnamic acid) gave a M+H peak at219 as expected. Further characterisation using ¹H (500 MHz) and ¹³C(125 MHz) NMR spectroscopy verified the structure.

c) Linking compound A to PEG(4)-N₃

To a solution of compound A (CP-471358, Pfizer, 41 mg, 87 μmol) in DMF(5 ml) were added 11-azido-3,6,9-trioxaundecanamine (19 mg, 87 μmol),HATU (Applied Biosystems, 33 mg, 87 μmol) and DIEA (Fluka, 30 μl, 174μmol). After one hour reaction time the mixture was concentrated and theresidue was purified by preparative HPLC (column Phenomenex Luna C18(2)5 μm 21.2×250 mm, solvents: A=water/0.1% TFA and B=acetonitrile/0.1%TFA; gradient 30-60% B over 60 min; flow 10.0 ml/min, UV detection at214 nm), giving 33.9 mg (59%) of product after lyophilisation. LC-MSanalysis (column Phenomenex Luna C18(2) 3 μm 50×4.60 mm, solvents:A=water/0.1% TFA and B=acetonitrile/0.1% TFA; gradient 20-100% B over 10min; flow 1 ml/min, UV detection at 214 nm, ESI-MS) gave a peak at 4.88min with m/z 667.4 (MH⁺) as expected.

d) Svnthesis of Compound A-PEG(4)-NH₂

To a solution of the PEG(4)-N₃ compound from c) (4.7 mg, 7.0 μmol) inmethanol (4 ml) was added Pd/C (Koch-Light, ca 10 mg) added. The mixturewas stirred at room temperature under hydrogen atmosphere (1 atm) for 10min. The mixture was filtered and concentrated. LC-MS analysis (columnPhenomenex Luna C18(2) 3 μm 50×4.60 mm, solvents: A=water/0.1% TFA andB=acetonitrile/0.1% TFA; gradient 20-100% B over 10 min; flow 1 ml/min,UV detection at 214 nm, ESI-MS) gave a peak at 4.17 min with m/z 641.4(MH⁺) as expected. The product was used directly in the next stepwithout further purification.

e) Conjugation of Cy 5.5

To a solution of the amine from d) (1.0 mg, 1.5 μmol) in DMF (0.2 ml)was added Cy 5.5-NHS (Amersham Biosciences, 1.0 mg, 1.0 μmol) andN-methylmorpholine (1 μl, 9 μmol). The reaction mixture was stirred for48 h. MS analysis of the solution gave a spectrum showing startingmaterial and the conjugated product at m/z 1539.7 (M⁺⁾, expected 1539.4.

1-12. (canceled)
 13. An optical imaging contrast agent with affinity foran abnormally expressed biological target associated with CRC, whereinthe contrast agent has a molecular weight below 10000 Daltons.
 14. Acontrast agent as claimed in claim 13 of formula IV-L-R   (I) wherein V is one or more vector moieties having affinity foran abnormally expressed target in CRC, L is a linker moiety or a bondand R is one ore more reporter moieties detectable in optical imaging.15. A contrast agent as claimed in claim 14 wherein R is a cyanine dye.16. A contrast agent as claimed in claim 13 wherein the target is areceptor or a non-catalytical target.
 17. A contrast agent as claimed inclaim 13 comprising a contrast agent substrate, wherein the target is anabnormally expressed enzyme, such that the contrast agent changespharmacodynamic properties and/or pharmacokinetic properties upon achemical modification from a contrast agent substrate to a contrastagent product upon a specific enzymatic transformation.
 18. A contrastagent as claimed in claim 17 wherein the contrast agent changes bindingproperties to specific tissue, membrane penetration properties, proteinbinding or solubility properties upon the chemical modification.
 19. Acontrast agent as claimed in claim 13 having affinity for any of thetargets selected from COX-2, beta-catenin, E-cadherin, P-cadherin,kinases, Her-2, MMPs, cyclins, P53, thymidylate synthase, VEGFreceptors, EGF receptors, K-ras, adenomatous polyposis coli protein,cathepsin B, uPAR, c-met, mucins and gastrin receptors.
 20. A contrastagent as claimed in claim 14 wherein V is selected from peptides,peptoid moieties, oligonucleotides, oligosaccharides, lipid-relatedcompounds and traditional organic drug-like small molecules.
 21. Acontrast agent as claimed in claim 20 wherein V is a peptide.
 22. Apharmaceutical composition for optical imaging for diagnosis of CRC, forfollow up of progress of CRC development or for follow up of treatmentof CRC, comprising a contrast agent as defined in claim 13 together withat least one pharmaceutically acceptable carrier or excipient.
 23. Acontrast agent as claimed in claim 13 for the manufacture of adiagnostic agent for use in a method of optical imaging of CRC involvingadministration of said diagnostic agent to an animate subject andgeneration of an image of at least part of said subject.
 24. A method ofgenerating an optical image of an animate subject involvingadministering a contrast agent to the subject and generating an opticalimage of at least a part of the subject to which the contrast agent hasdistributed, characterized in that a contrast agent as defined in claim13 is used.